The computational power of the brain depends on synaptic connections that link together billions of neurons. With the long-term goal of understanding how synaptic signaling regulates neuronal communication and connectivity, and how its dysfunction contributes to neurological disease, we propose to use Drosophila as a model system for determining the molecular mechanisms underlying neurotransmitter release and retrograde signaling. Despite the differences in complexity between flies and mammals, genomic analysis suggests many key neuronal proteins and the functional mechanisms they govern are remarkably similar. In Aim 1, we propose to elucidate the underlying molecular interactions mediated by the SNARE binding proteins Synaptotagmin 1 and Complexin that mediate rapid information transfer from the presynaptic terminal. We have generated screening approaches that allow F1 genetic screens for Synaptotagmin 1 point mutants, allowing us to do saturation mutagenesis to identify key residues and protein interactions that are required for the regulation of synaptic vesicle fusion. In addition we will determine how the Ca2+ sensor Synaptotagmin 1 and the synaptic vesicle fusion clamp Complexin coordinately regulate information transfer from the presynaptic terminal. These studies will generate new mechanistic insights into the synaptic vesicle fusion machinery that will go well beyond our current understanding of neurotransmitter release. In Aim 2, we propose to define the molecular machinery for Ca2+-regulated postsynaptic release that mediates retrograde signaling. Unlike the synaptic vesicle-associated Synaptotagmin 1, we found that Synaptotagmin 4 is expressed postsynaptically and mutants lacking Synaptotagmin 4 show abnormal development and function of synapses. Our genetic analysis of synaptic plasticity in Drosophila suggests that Ca2+-dependent retrograde vesicular trafficking mediated by Synaptotagmin 4 initiates an acute change in synaptic function that is converted to synapse- specific growth, providing a link between synaptic plasticity and activity-dependent rewiring of neuronal connections. We will characterize this retrograde signaling pathway by identifying additional components of the postsynaptic vesicle fusion machinery and the retrograde signals involved. We will also compare the functional properties of the two Synaptotagmin isoforms in mediating pre- versus post-synaptic vesicle fusion. The proposed studies will provide important insights into how the nervous system functions at the cellular level, allowing us to integrate this information into the framework of ultimately understanding how neuronal ensembles mediate behavior, and how neurological and psychiatric diseases disrupt these processes.